Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules

As the interest in DNA nanotechnology increases, so does the need for larger and more complex DNA structures. In this work, we describe two methods of using large, double-stranded (ds) DNA to self-assemble sequence-specific, nonrepetitive microscale structures. A model system restructures T7 DNA (40 kb) through sequence-specific biotinylation followed by intramolecular binding to a 40 nm diameter neutravidin bead to create T7 “rosettes”. This model system informed the creation of “nodal DNA” where “nodes” with single-stranded DNA flaps are attached to a large dsDNA insert so that a complementary oligonucleotide “strap” bridges the two nodes for restructuring to form a DNA “bolo”. To do this in high yield, several methodologies were developed, including a protection/deprotection scheme using RNA/RNase H and dialysis chambers, which remove excess straps while retaining large DNA molecules. To assess these restructuring processes, the DNA was adsorbed onto supported lipid bilayers, allowing for a visual assay of their structure using single-molecule fluorescence microscopy. Good agreement between the expected and observed fluorescence intensity measurements of the individual features of restructured DNA for both the DNA rosettes and bolos gives us a high degree of confidence that both processes give sequence-specific restructuring of large, dsDNA molecules to create microscale objects.